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Preface | p. xiii |
Acknowledgements | p. xv |
Abbreviations and acronyms | p. xvii |
DNA: Structure and function | p. 1 |
Nucleic acid is the material of heredity | p. 2 |
Structure of nucleic acids | p. 7 |
The double helix | p. 11 |
The antiparallel helix | p. 12 |
Base pairs and stacking | p. 14 |
Gaining access to information with the double helix without breaking it apart | p. 16 |
Hydrogen bonding | p. 17 |
Reversible denaturing of DNA | p. 18 |
Structure of DNA in the cell | p. 21 |
The eukaryotic nucleosome | p. 24 |
The replication of DNA | p. 28 |
DNA polymerases | p. 31 |
The replication process | p. 33 |
Recombination | p. 37 |
Genes and genomes | p. 39 |
Genes within a genome | p. 40 |
Transcription | p. 43 |
Transcription in prokaryotes | p. 43 |
Transcription in eukaryotes | p. 46 |
RNA processing | p. 54 |
RNA splicing | p. 55 |
Alternative splicing | p. 58 |
Translation | p. 59 |
Basic techniques in gene analysis | p. 65 |
Restriction enzymes | p. 66 |
Types of restriction-modification system | p. 70 |
Other modification systems | p. 72 |
How do type II restriction enzymes work? | p. 74 |
Joining DNA molecules | p. 76 |
The basics of cloning | p. 78 |
Bacterial transformation | p. 84 |
Chemical transformation | p. 86 |
Electroporation | p. 87 |
Gene gun | p. 88 |
Gel electrophoresis | p. 88 |
Polyacrylamide gels | p. 89 |
Agarose gels | p. 89 |
Pulsed-field gel electrophoresis | p. 95 |
Nucleic acid blotting | p. 98 |
Southern blotting | p. 100 |
The compass points of blotting | p. 102 |
DNA purification | p. 103 |
Vectors | p. 109 |
Plasmids | p. 112 |
pBR322 | p. 116 |
pUC plasmids | p. 119 |
Selectable markers | p. 122 |
[lambda] vectors | p. 126 |
Cosmid vectors | p. 135 |
M13 vectors | p. 137 |
Phagemids | p. 140 |
Artificial chromosomes | p. 142 |
YACs | p. 143 |
PACs | p. 146 |
BACs | p. 148 |
HACs | p. 149 |
Polymerase chain reaction | p. 153 |
PCR reaction conditions | p. 159 |
Thermostable DNA polymerases | p. 162 |
Template DNA | p. 164 |
Oligonucleotide primers | p. 165 |
Synthesis of oligonucleotide primers | p. 167 |
Primer mismatches | p. 169 |
PCR in the diagnosis of genetic disease | p. 173 |
Cloning PCR products | p. 175 |
RT-PCR | p. 177 |
Real-time PCR | p. 179 |
Applications of PCR | p. 181 |
Cloning a gene | p. 183 |
Genomic libraries | p. 185 |
cDNA libraries | p. 191 |
Directional cDNA cloning | p. 196 |
PCR based libraries | p. 199 |
Subtraction libraries | p. 200 |
Library construction in the post-genome era | p. 204 |
Gene identification | p. 205 |
Screening by nucleic acid hybridization | p. 206 |
Immunoscreening | p. 211 |
Screening by function | p. 216 |
Screening by interaction | p. 217 |
Phage display | p. 218 |
Two-hybrid screening | p. 218 |
Problems, and some solutions, with two-hybrid screening | p. 225 |
Other interaction screens--variations on a theme | p. 228 |
One hybrid | p. 229 |
Three hybrid | p. 229 |
Reverse two hybrid | p. 229 |
Creating mutations | p. 231 |
Creating specific DNA changes using primer extension mutagenesis | p. 233 |
Strand selection methods | p. 237 |
Phosphorothioate strand selection | p. 237 |
dut[superscript -] ung[superscript -] (or Kunkel) strand selection | p. 238 |
Cassette mutagenesis | p. 240 |
PCR based mutagenesis | p. 241 |
QuikChange mutagenesis | p. 248 |
Creating random mutations in specific genes | p. 250 |
Protein engineering | p. 254 |
Protein production and purification | p. 257 |
Expression in E. coli | p. 258 |
The lac promoter | p. 259 |
The tac promoter | p. 259 |
The [lambda]P[subscript L] promoter | p. 260 |
The T7 expression system | p. 261 |
Expression in yeast | p. 265 |
Saccharomyces cerevisiae | p. 265 |
The GAL system | p. 266 |
The CUP1 system | p. 268 |
Pichia pastoris | p. 268 |
Schizosaccharomyces pombe | p. 269 |
Expression in insect cells | p. 269 |
Expression in higher-Eukaryotic cells | p. 272 |
Tet-on/Tet-off system | p. 272 |
Protein purification | p. 275 |
The His-tag | p. 276 |
The GST-tag | p. 279 |
The MBP-tag | p. 282 |
IMPACT | p. 282 |
TAP-tagging | p. 286 |
Genome sequencing projects | p. 287 |
Genomic mapping | p. 289 |
Genetic mapping | p. 290 |
Physical mapping | p. 293 |
Nucleotide sequencing | p. 295 |
Manual DNA sequencing | p. 296 |
Automated DNA sequencing | p. 300 |
Genome sequencing | p. 303 |
The human genome project | p. 305 |
Finding genes | p. 307 |
Gene assignment | p. 309 |
Bioinformatics | p. 311 |
Post-genome analysis | p. 313 |
Global changes in gene expression | p. 314 |
Differential display | p. 315 |
Microarrays | p. 317 |
ChIPs with everything | p. 324 |
Protein function on a genome-wide scale | p. 327 |
Knock-out analysis | p. 327 |
Antisense and RNA interference (RNAi) | p. 329 |
Genome-wide two-hybrid screens | p. 333 |
Protein detection arrays | p. 335 |
Structural genomics | p. 335 |
Engineering plants | p. 341 |
Cloning in plants | p. 341 |
Agrobacterium tumefaciens | p. 342 |
Direct nuclear transformation | p. 347 |
Viral vectors | p. 348 |
Chloroplast transformation | p. 350 |
Commercial exploitation of plant transgenics | p. 354 |
Delayed ripening | p. 354 |
Insecticidal resistance | p. 355 |
Herbicidal resistance | p. 356 |
Viral resistance | p. 357 |
Fungal resistance | p. 358 |
Terminator technology | p. 358 |
Ethics of genetically engineered crops | p. 360 |
Engineering animal cells | p. 361 |
Cell culture | p. 361 |
Transfection of animal cells | p. 362 |
Chemical transfection | p. 363 |
Electroporation | p. 364 |
Liposome-mediated transfection | p. 364 |
Peptides | p. 366 |
Direct DNA transfer | p. 366 |
Viruses as vectors | p. 367 |
SV40 | p. 367 |
Adenovirus | p. 369 |
Adeno-associated virus (AAV) | p. 371 |
Retrovirus | p. 372 |
Selectable markers and gene amplification in animal cells | p. 375 |
Expressing genes in animal cells | p. 378 |
Engineering animals | p. 379 |
Pronuclear injection | p. 381 |
Embryonic stem cells | p. 384 |
Nuclear transfer | p. 390 |
Gene therapy | p. 396 |
Examples and potential of gene therapy | p. 398 |
Glossary | p. 401 |
Proteins | p. 409 |
p. 409 | |
p. 410 | |
p. 411 | |
Nobel prize winners | p. 413 |
References | p. 417 |
Index | p. 459 |
Table of Contents provided by Rittenhouse. All Rights Reserved. |
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"This beautifully illustrated textbook provides a clear guide to the tools and techniques of genetic engineering, gene cloning and molecular biology. All aspects of genetic engineering in the post-genomic era are covered, beginning with the basics of DNA structure and DNA metabolism. Using an example-driven approach, the fundamentals of creating mutations in DNA, cloning in bacteria, yeast, plants and animals are all clearly presented. Strong emphasis is placed on the latest, post genomic technologies including DNA macro and microarrays, genome-wide two hybrid analysis, proteomics and bioinformatics. A modern post-genome era introduction to key techniques used in genetic engineering. An example driven past-to-present approach to allow the experiments of today to be placed in an historical context The book is beautifully illustrated in full-colour throughout. Associated website including updates, additional content and illusions "
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